Hepatitis A virus deletion mutants and vaccine formulations containing the same

ABSTRACT

Disclosed are live hepatitis A virus deletion mutants having a deletion mutation in the 5&#39; nontranslated region of the viral genome. The deletion mutation may be selected from the group consisting of (a) pY1 deletion mutations that cause the virus to retain the ability to replicate in monkey kidney cells; and (b) deletion mutations between nucleotides 140 and 144 that render the virus temperature sensitive. Advantageously, the deletion mutation may be an attenuating mutation. Pharmaceutical formulations containing such viruses are disclosed, along with the use thereof to produce antibodies useful for diagnostic purposes and for imparting protective immunity against hepatitis A virus.

This invention was made with government support under grants RO1-AI32599and T32-AI07001 from the US Public Health Service, and by grant DAMD17-89-Z-9022 from the US Army Medical Research and Development Command.The government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to hepatitis A virus containing deletionmutations in the 5' nontranslated region thereof, and live attenuatedvaccine formulations containing the same.

BACKGROUND OF THE INVENTION

Like other members of the picornavirus family, hepatitis A virus (HAV,genus hepatovirus) contains a single-stranded, positive-sense RNA genomewith a lengthy (735 nucleotide nt!) 5' nontranslated region (5'NTR). The5'NTR of HAV (HM175 strain) contains an internal ribosomal entry site(IRES) located between nt 152 and nt 735, which regulatescap-independent, internal initiation of translation of the viralpolyprotein, although the level of efficiency with which the HAV IRESpromotes translation is much less than that of other picornavirus IRESelements both in vivo and in vitro (E. Brown, A. Zajac, and S. Lemon, J.Virol. 68:1066-1074 (1994), L. Whetter, et al., J. Virol. In press(1994)). The 5'NTR of HAV may possibly have other functions which areessential for virus replication, including control of positive-strandRNA synthesis and possibly encapsidation (R. Andino et al., Cell63:369-380 (1990)).

Although there are substantial differences between the predictedsecondary structures of the 5'NTRs of HAV and all other picornaviruses,the secondary structure of the HAV 5'NTR more closely resembles that ofthe cardioviruses and aphthoviruses than the corresponding structure inrhinoviruses and most enteroviruses (E. Brown, et al., J. Virol.65:5828-5838 (1991)). Among other similarities, the 5'NTRs ofhepatoviruses, cardioviruses, and aphthoviruses share the potential toform two or more pseudoknots in the noncoding region upstream of theIRES element (E. Brown, et al., J. Virol. 65:5828-5838 (1991), E. Brown,A. Zajac, and S. Lemon, J. Virol. 68:1066-1074 (1994), B. Clarke, etal., Nucleic Acids Res. 15:7066-7079 (1987), S. Jang, et al., J. Virol.63:1651-1660 (1989), R. Kuhn, N. Luz, and E. Beck, J. Virol.64:4625-4631 (1990), C. Pleij, Proc. VIII Int. Congr. Virol. 49-50(1990)). Also present in this region is a pyrimidine-rich sequence whichconsists of an almost pure polycytidylic acid tract in the cardiovirusesand aphthoviruses. In the hepatoviruses, the corresponding regioncontains a mixture of uridylic acids and cytidylic acids (in a 24:14ratio in the HM175 strain of HAV), with only two purines located withina 40-nt-long, nearly pure polypyrimidine tract (pY1 domain, nt 99 to138). In each of these virus genera, this pyrimidine-rich tract appearsto separate two discrete regions of RNA secondary structure (E. Brown,et al., J. Virol. 65:5828-5838 (1991), B. Clarke, et al., Nucleic AcidsRes. 15:7066-7079 (1987)). A similarly located pyrimidine-rich tract isnot found in either the enterovirus or rhinovirus 5'NTR.

There are other pyrimidine-rich tracts within the 5'NTR of HAV (K.Chang, E. Brown, and S. Lemon, J. Virol. 67:6716-6725 (1993)), but thepY1 domain is the lengthiest and most prominent of these regions.Although considerable sequence heterogeneity exists within this domainamong different human hepatoviruses (E. Brown, et al., J. Virol.65:5828-5838 (1991)), the general features of this domain are conservedamong all strains of HAV. A striking aspect of the pY1 domain, which isunique to the HAV 5'NTR among all other picornaviral 5'NTRs, is thepresence of tandem repeats of the sequence motif (U)UUCC(C). Curiously,this motif closely resembles the core sequence of the "box A" motif ofPilipenko et al. (E. Pilipenko, et al., Cell 68:119-131 (1992)), whichis present in a conserved location in all picornaviruses, about 20-25 ntupstream of the initiator AUG and which may play an important role ininternal initiation of translation.

Previous modeling of the secondary structure of the 5'NTR of HM175 viruspredicted that the pY1 domain and the immediately adjacent sequence fromnt 139 to nt 154 were likely to be single-stranded (E. Brown, et al., J.Virol. 65:5828-5838 (1991)).

SUMMARY OF THE INVENTION

Here, we present the results of RNase mapping of the secondary structureof the pY1 region of the HM175 5'NTR, and describe a series of mutantviruses with large deletions involving the pY1 domain. We show that thepY1 domain forms an ordered structural element downstream of theputative 5' pseudoknots of HAV, but that this ordered structure is notrequired for efficient replication in cultured cells as long as thesequence between nt 140 and nt 144 is present. In contrast, an extendedsingle-stranded region immediately downstream of the pY1 domain, whichincludes nt 140 to nt 144, is required for efficient replication of thevirus at physiologic temperatures.

Further disclosed herein is the attenuating nature of the foregoingmutations and their use in the preparation of live hepatitis A virusvaccines.

A first aspect of the present invention is a live hepatitis A virusdeletion mutant having a deletion mutation in the 5' nontranslatedregion of the viral genome. The deletion mutation may be selected fromthe group consisting of (a) pY1 deletion mutations that cause the virusto retain the ability to replicate in monkey kidney cells; and (b)deletion mutations between nucleotides 140 and 144 that render the virustemperature sensitive. Advantageously, the deletion mutation may be anattenuating mutation. Examples include (i) the deletion of at least 6nucleotides between nucleotide 94 and nucleotide 140, inclusive (e.g.,deletion mutations such that the pY1 region of the viral genome is notmore than 20 nucleotides in length), and (ii) the deletion of at leastone nucleotide between nucleotides 141 and 144, inclusive.

A second aspect of the present invention is a cDNA encoding a virus asgiven above.

A third aspect of the present invention is a method for inducingprotective immunity against hepatitis A virus. The method comprisesadministering to a subject an infectious, immunogenic, hepatitis A viruscarrying an attenuating mutation as given above, the virus beingadministered in an amount effective to induce protective immunityagainst hepatitis A virus. Preferably, administration is by oraladministration.

A fourth aspect of the present invention is the use of an infectious,immunogenic, hepatitis A virus as given above for the preparation of amedicament for imparting protective immunity against hepatitis A virusto a subject.

A fifth aspect of the present invention is a vaccine formulation usefulfor inducing protective immunity against hepatitis A virus, comprising,an infectious, immunogenic, hepatitis A virus carrying an attenuatingmutation, as given above, in a pharmaceutically acceptable carrier.

The foregoing and other objects and aspects of the present invention areexplained in detail in the drawings herein and the specification setforth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows pY1 deletion mutations constructed within a full-lengthcDNA clone of the HM175 virus. The wild-type virus (SEQ. ID NO. 10)sequence is shown at the top, with mutant plasmids listed at the left(SEQ ID NO. 11-24). Boxes represent nucleotides predicted to be involvedin base-pairing interactions in stem-loops IIb (nt 90 to 94) or IIIa (nt155 to 159); Dots represent positions cleaved by single-strand specificRNases. Direct transfection results at 31° C. and 37° C. are shown atthe right: (+) replication foci demonstrated; (-) no replication focidemonstrated; n.d., not determined. Nucleotides 140 to 144 which aredeleted in ts viruses are indicated at the bottom.

FIG. 2A shows intracellular virus accumulation under one step growthconditions at 31° C. or 37° C. for the P16-pY1 virus. BS-C-1 cells wereinfected with a virus inoculum calculated to provide a multiplicity ofinfection of approximately 3 infectious particles per cell and thenincubated at either 31° C. (∘) or 37° C. (▪). At the indicated timepoints, monolayers were washed and lysed by the addition of 0.1% SDS.Virus titers in lysates were determined by radioimmunofocus assay inBS-C-1 cells at 31° C.

FIG. 2B shows intracellular virus accumulation under one step growthconditions at 31° C. or 37° C. for 96-137 virus. Conditions are the sameas for FIG. 2A.

FIG. 2C shows intracellular virus accumulation under one step growthconditions at 31° C. or 37° C. for 131-144 virus. Conditions are thesame as for FIG. 2A.

FIG. 3 shows the thermostability of the P16-pY1 (□) and ts Δ131-144 ()viruses. Virus stocks were incubated at the indicated temperature for 10minutes, and the surviving virus titer determined by radioimmunofocusassay of BS-C-1 cells at 31° C.

FIG. 4 shows the alignment of 13 human hepatitis A virus strains betweennt 90 and nt 155 (SEQ ID NO. 25-37) (HM175/wt sequence numbering).Strains marked by (*) were sequenced after passage in cell culture,while others were sequenced directly from primate materials. Nucleotidesflanking the region of interest which are conserved among all strainsare shown in boldface type. Cytidylic acid residues within the(U)UUCC(C) motifs are indicated by a double underline. The preciseorigin of PA21 virus is obscure: this virus was isolated from anaturally infected captive owl monkey, but viruses with very similarnucleotide sequences have been recovered from humans (R. Jansen, G.Siegl, and S. Lemon, Proc. Natl. Acad. Sci. USA 87:2867-2871 (1990)).The sequence of Ken virus (SEQ ID NO. 34) was determined from viruswhich had been passaged once in chimpanzees. Sequences of AZ79, (SEQ IDNO. 26) Chi81, (SEQ ID NO. 27) Ita85, (SEQ ID NO. 28) Ga88, (SEQ ID NO.29) and Ken (SEQ ID NO. 34) were provided by Dr. B. Robertson, Centersfor Disease Control and Prevention, Atlanta. The sequence from strain CP(SEQ ID NO. 30) was determined during the course of this work. For othersequences, see (E. Brown, et al., J. Virol. 65:5828-5838 (1991)).

DETAILED DESCRIPTION OF THE INVENTION

The numbering of nucleotides herein is given with reference to hepatitisA virus strain HM175. However, the instant invention is applicable toany hepatitis A virus strain. Numbering of nucleotides in strains ofhepatitis A virus other than strain HM175 is accordingly carried out byalignment of other strains to HM175 for maximum homology within theregions flanking the pY1 region in the 5' and the 3' direction, and thenapplying the numbering system for strain HM175 to these other strainswithin the pY1 region. Since some hepatitis A virus strains contain pY1regions shorter than HM175, pY1 regions missing or nut present underthis numbering system are considered to be deleted. An example of suchan alignment and numbering is shown in FIG. 4 (discussed below).

Viruses used to carry out the present invention for the purpose ofmaking vaccine formulations, immunizing subjects, or making antibodiesare live viruses that are immunogenic (i.e., produce an immune responseto hepatitis A virus in a subject). The viruses may be viruses that arevirulent but for the presence of the attenuating mutations describedherein (the term "virulent" meaning capable of causing disease in aninfected subject). Illustrative strains of hepatitis A virus used tocarry out the present invention include, but are not limited to, strainHM175, strain CR326, strain MBB, strain GBM, etc.

Attenuating mutations to the pY1 region are preferably betweennucleotides 94 and nucleotide 140 (since the pY1 region begins atnucleotide 99 and ends at nucleotide 138, it will be appreciated thatthe deletion may begin 5' to the pY1 region and end 3' to the pY1region). The attenuating mutation is preferably at least 6 nucleotidesin length (i.e., at least 6 are deleted), and may be 8, 10, 12, 14, 16,18, 20, 22, 24, 26, nucleotides in length or more, up to deletion of theentire pY1 region. Larger deletions are preferred. Stated otherwise,viruses containing attenuating mutations of the instant invention have apY1 region of not more than 20, 18, 16, 14, 12, 10, 8, 6, 4, or 2nucleotides in length, total, up to the complete deletion (i.e. completeabsence) of the pY1 region. Examples of hepatitis A viruses containingsuch deletion mutations are:

(a) Δ99-115 deletion mutants;

(b) Δ99-130 deletion mutants;

(c) Δ99-134 deletion mutants;

(d) Δ96-134 deletion mutants;

(e) Δ96-137 deletion mutants;

(f) Δ96-139 deletion mutants; and

(g) Δ96-140 deletion mutants.

The foregoing attenuating mutations may be combined with otherattenuating mutations, such as deletion mutations to the 3' flankingregion, as discussed below.

Attenuating mutations may also be in the 3' flanking region of the pY1region (i.e., nucleotides 141-144 in the HM175 genome or other strainswhen numbered with reference to the HM175 genome; also sometimesdesignated nucleotides numbers 1-4 herein for convenience). Suchattenuating mutations may be one, two, three, or four nucleotides inlength. It is preferred that at least one, and more preferred that atleast two, of nucleotides 142, 143, and 144 (or nucleotides 2, 3, and 4)be deleted. Most preferably at least all three of nucleotides 142 to 144are deleted. Examples of hepatitis A viruses containing such deletionmutations are:

(h) Δ142 deletion mutants;

(i) Δ143 deletion mutants;

(j) Δ144 deletion mutants;

(k) Δ141-142 deletion mutants;

(l) Δ142-143 deletion mutants;

(m) Δ143-144 deletion mutants;

(n) Δ141-143 deletion mutants;

(o) Δ143-144 deletion mutants; and

(p) Δ140-144 deletion mutants.

The foregoing deletion mutations may be combined with other mutations(e.g., as all are in a Δ141-144 deletion mutant), including deletionmutations to the pY1 region as discussed above and below.

Attenuating mutations may be in both the pY1 region and the 3' flankingregion thereof, in combination. Where the attenuating mutation is inboth regions, the attenuating mutation may be either continuous ordiscontinuous. An example of a discontinuous attenuating mutation wouldbe a deletion of all but 10 nucleotides of the pY1 region and thedeletion of all but nucleotide 141 of the 3' flanking region. Examplesof hepatitis A viruses containing such deletion mutations as continuousmutations are:

(q) Δ96-141 deletion mutants;

(r) Δ99-144 deletion mutants;

(s) Δ116-144 deletion mutants; and

(t) Δ131-144 deletion mutants.

The foregoing deletion mutations may optionally be combined with one ormore other mutation(s), including other attenuating mutations, and/orother mutations that promote growth in cultured mammalian cells,elsewhere in the viral genome. Other such mutations are not critical solong as the virus is live, infectious and immunogenic. Examples of suchother mutations include, but are not limited to, those described in U.S.Pat. No. 4,894,228 to Purcell et al. (the disclosure of all patentreferences cited herein is to be incorporated herein by reference). Inaddition, minor substitutions mutations may also be made within oradjacent to the deletion mutations herein described, as will beappreciated by those skilled in the art.

Attenuating mutations of the instant invention are introduced into cDNAsencoding live, infectious, hepatitis A virus by any suitable means, suchas by PCR mutagenesis (discussed below) and by site-directed mutagenesis(see, e.g., U.S. Pat. No. 4,873,192 to Kunkel).

Virus carrying mutations of the present invention are made byconventional means. In general, a cDNA encoding live virus carrying thedesired attenuating mutation is introduced into a cell line and the cellline cultured to produce live virus in the culture. Where the virus isto be used as a vaccine, the cell line is typically a continuousmammalian cell line that is certified for use in the production of humanor veterinary vaccines (e.g., MMC5 cells, VERO cells, etc.). Once livevirus containing the attenuating mutation is obtained, a seed stock ofthe RNA virus can be established and infected cells used to initiate newcultures without the need for introducing a cDNA into the cells. In themanufacture of a pharmaceutical formulation, virus is collected from theculture and combined with a pharmaceutically acceptable carrier, asdiscussed in greater detail below.

Oral vaccine formulations may be made from a culture of cells producinglive virus containing the desired deletions in accordance with knowntechniques. The culture itself may be administered to the subject; theculture may be optionally filtered and/or clarified; stabilizers such assucrose, MgCl₂, etc. may be added to the media.

Exemplary pharmaceutically acceptable carriers include, but are notlimited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution. Pharmaceutically acceptable carriers fororal administration may be a syrup, elixir, lozenge, etc. The vaccineformulation may be prepared in accordance with known techniques, such asillustrated by R. Purcell et al., Vaccine Against Hepatitis A Virus,U.S. Pat. No. 4,894,228.

Subjects which may be administered the live attenuated viruses andvaccine formulations disclosed herein include both human subjects andanimal subjects (e.g., the veterinary treatment of primates such as owlmonkeys, marmosets and chimpanzees).

Vaccine formulations of the present invention comprise an immunogenicamount of a live attenuated virus as disclosed herein in combinationwith a pharmaceutically acceptable carrier. An "immunogenic amount" isan amount of the attenuated virus sufficient to evoke an immune responsein the subject to which the virus is administered. The particular doseemployed is not critical, and depends upon the type and condition of thesubject, the route of administration, etc. An amount of from about 10⁴to 10⁷ radioimmunofocus forming units of the live virus per dose istypically suitable (titration of radioimmunofocus forming units is asdescribed in S. Lemon et al., J. Clin. Microbiol. 17, 834-839 (1983).

Administration of the live attenuated viruses disclosed herein may becarried out by any suitable means, including both parenteral injection(such as intraperitoneal, subcutaneous, or intramuscular injection), byoral administration, and by topical application of the virus (typicallycarried in the pharmaceutical formulation) to an airway surface. Topicalapplication of the virus to an airway surface can be carried out byintranasal administration (e.g., by use of a dropper, swab, or inhalerwhich deposits a pharmaceutical formulation intranasally). Topicalapplication of the virus to an airway surface can also be carried out byinhalation administration, such as by creating respirable particles of apharmaceutical formulation (including both solid particles and liquidparticles) containing the virus as an aerosol suspension, and thencausing the subject to inhale the respirable particles. Methods andapparatus for administering respirable particles of pharmaceuticalformulations are well known, and any conventional technique can beemployed. See, e.g., U.S. Pat. No. 5,304,125 to D. Leith; U.S. Pat. No.5,299,566 to C. Davis and R. Snyder; U.S. Pat. No. 5,290,550 to R.Fisher and W. Metzger; and U.S. Pat. No. 5,292,498 to R. Boucher.

While the viruses, methods and formulations of the present inventionhave been described above with reference to producing protectiveimmunity, they may also be used to simply produce antibodies in animals,which antibodies may be used for the purpose of detecting and diagnosinghepatitis A virus in accordance with conventional diagnostic techniques.

The present invention is explained in greater detail in the examples setforth in the following experimental section.

EXPERIMENTAL

I. MATERIALS AND METHODS

Cells. HAV was propagated in continuous African green monkey kidney(BS-C-1) or fetal rhesus kidney (FRhK-4) cells as previously described(L. Binn, et al., J. Clin. Microbiol. 20:28-33 (1984)).

HAV cDNA plasmids. Deletion mutations were constructed within aninfectious, full length cDNA clone of the HM175 strain of HAV (FIG. 1).The parental clone was a chimeric infectious cDNA, pG7/18fP2, which wasconstructed by replacing the small SacI/EcoRI fragment of pG3/HAV7 (J.Cohen, et al., J. Virol. 61:3035-3039 (1987), S. Day, et al., J. Virol.66:6533-6540 (1992)) (HM175/P35 virus sequence) with the correspondingcDNA fragment from a rapidly replicating, cytolytic variant (HM175/18fvirus) (S. Lemon, et al., J. Virol. 65:2056-2065 (1991)), essentiallyreplacing the P2 region of HM175/P35 with that of HM175/18f virus (S.Lemon, et al., J. Virol. 65:2056-2065 (1991)). Transfections withpG7/18fP2 RNA give rise to visible, macroscopic replication foci inradioimmunofocus assays (S. Lemon, L. Binn, and R. Marchwicki, J. Clin.Microbiol. 17:834-839 (1983)) within 7 to 10 days, while transfectionswith pG3/HAV7 RNA generally require 14 to 21 days. Because the 5'NTR ofpG7/18fP2 is derived from HM175/P35 virus (J. Cohen, et al., Proc. Natl.Acad. Sci. USA 84:2497-2501 (1987)), we refer to this plasmid aspP35-pY1 in this communication. Compared with the wild type sequence,pP35-pY1 contains a 4-nt deletion (nt 131 to 134) and a single pointmutation (nt 124, U to C) within the pY1 domain (J. Cohen, et al., Proc.Natl. Acad. Sci. USA 84:2497-2501 (1987)). For consistency, allnucleotide numbering is according to the wild-type HM175 virus sequence(J. Cohen, et al., J. Virol. 61:50-59 (1987)). Except where noted,manipulations of the pY1 domain were carried out in pB1.O, whichcontains a cDNA copy of the first 2024 bases of the HM175/P16 (ratherthan HM175/P35) virus sequence (E. Brown, A. Zajac, and S. Lemon, J.Virol. 68:1066-1074 (1994), R. Jansen, J. Newbold, and S. Lemon,Virology 163:299-307 (1988)). The pY1 domain of HM175/P16 is identicalto that of the wild type virus. Outside of the pY1 domain, the sequenceof the P16 and P35 variants of HM175 differ at only a single baseposition in the region manipulated during mutagenesis (nt 25 to 632, seebelow). We have shown previously that these two 5'NTR sequences arefunctionally identical with respect to their ability to support viralreplication in cultured cells (S. Day, et al., J. Virol. 66:6533-6540(1992)).

Several different mutagenesis strategies were employed in constructingthe deletion mutants shown in FIG. 1. The initial strategy involvedconstruction of a subclone with unique restriction sites flanking thepY1 domain. Mutagenic oligonucleotide primers (TTTGCCTAGGCTATAGGCTCCATTpositive sense!) (SEQ ID NO. 1) and (TGAACCTGCAGGAACCAATATTTA negativesense!) (SEQ ID NO. 2) were used to amplify the region between bases 78and 168 of pB1.O by polymerase chain reaction (PCR) and to create NlaIVsites immediately downstream of the second predicted pseudoknot ofdomain II (stem loop IIb, nt 95) and immediately upstream of the firstpredicted stem loop of domain III (IIIa, nt 154) (not shown) (E. Brown,et al., J. Virol. 65:5828-5838 (1991)). The resulting 90 base PCRproduct containing the NlaIV sites was gel-purified and used as thenegative strand primer in a second PCR reaction, in which the positivestrand primer began at the -7 position relative to the HAV sequence andincluded the HindIII site at which the HAV insert is cloned in thevector. The 0.17 kb product of the second PCR reaction was digested withHindIII and PstI, and then ligated with a PstI/BamHI fragment of pB1.O(nt 162 to 632), into HindIII and BamHI sites of pGEM3zf(-) (Promega) tocreate pG3zNla. This clone contains the first 632 bases of the HM175/P16sequence with new NlaIV sites at nt 95 and nt 154. Several clones withspecific mutations within the pY1 domain were constructed by ligation ofblunt-ended inserts into pG3zNla following digestion with NlaIV andremoval of the region spanning nt 95 to 154. The three base changeswhich created the new NlaIV sites in PG3zNla were at positions 96, 97,and 153, and were thus removed prior to the ligations.

A mutant insert containing a 46-base deletion between nt 99 and nt 144was created by annealing the two complementary oligonucleotides(TAAAAAATATTGAT positive sense!) (SEQ ID NO. 3) and (ATCAATATTTTTTAnegative sense!) (SEQ ID NO. 4), and ligating the duplex into pG3zNla.The resulting subclone, containing the correct HAV sequence (HM 175/pl6)from 0 to 632 except for the deletion, was used to create a full-lengthcDNA clone, pΔ99-144 (deletion spanning nt 99 to 144) (FIG. 1), byligating the 0.61 kb BspEI/BamHI fragment (HAV sequence between bases 25and 632) with the 9.7 kb fragment of pP35-pY1 resulting from BspEI andpartial BamHI digestion. As a control for these manipulations, an insertwhich contained the sequence of HM175/pl6 virus spanning nt 96 to 155(HM175/P16 numbering) was created using pB1.0 as template in a PCR. Thissequence was similarly introduced into pP35-pY1 to create pPl6-pY1 ,which contains the HM175/P16 (or HM175/wt) sequence within the pY1domain.

PCR mutagenesis was subsequently used to create a series of full-lengthcDNA clones with progressive deletions from the 5' or 3' end of thepyrimidine-rich tract. Fifty to 60 base long negative-sense primerscontaining the PstI site at nt 162 were used to add back either 14 or 17bases of the deleted pyrimidine-rich sequence to the PCR template,pΔ99-144, which lacked the entire pY1 domain. The positive sense primerin each of these reactions spanned the BspEI site at nt 25. Theresulting PCR products were digested with BspEI and PstI, and ligatedinto the unique BspEI/PstI site of a subclone containing the 0.63 kbHindIII/BamHI fragment of HAV (HM175/P16 sequence). Finally, a 0.61 kbproduct derived by digestion of this intermediate subclone withBspEI/BamHI was ligated with the 9.7 kb BspEI/BamHI fragment of pP35-pY1as above. Clones obtained in this manner included pΔ99-130 and pΔ116-144(FIG. 1). A similar strategy, using pΔ99-130 and pΔ116-144 as templatesfor PCR mutagenesis, was taken to add back or delete additionalnucleotides creating pΔ99-115, pΔ99-134 and pΔ131-144. dsDNA sequencingconfirmed the fidelity of PCR amplified segments and the mutationspresent in individual cDNA clones.

Additional full-length mutant clones (pΔ96-134, pΔ93-134, pΔ96-137,pΔ96-139, pΔ96-140, and pΔ96-141) (FIG. 1) were created by heteroduplexsite-directed mutagenesis (S. Inouye and M. Inouye, Academic Press,Orlando 181-206 (1987)), using pΔ99-134, pΔ96-134, or pΔ96-137 astemplates, without construction of the intermediate subclone. Templatesequence between the BspEI site (nt 25) and the HpaI site (nt 353) wasmade single-stranded in heteroduplexes formed by DNA which had beendigested with BglI (first strand) or BspEI/HpaI (second strand). Amutagenic oligonucleotide primer was annealed to this region, thesingle-stranded regions of the heteroduplexes were filled in by theKlenow fragment of DNA polymerase I, and the ends ligated by T4 DNAligase. All cDNA clones made by heteroduplex site-directed mutagenesiswere sequenced between the BspEI and HpaI sites.

Physical mapping of RNA secondary structure. RNAs representing the first1 kb of the HAV genome were synthesized as runoff transcripts from DNAclones linearized at the XmnI site (nt 980) or the NdeI site (nt 1108).Reaction products were digested with DNAse, extracted withphenol-chloroform, and ethanol precipitated. The RNA transcripts wereheated to 65° C. for 3 minutes and allowed to cool slowly to 4° C. inthe presence of 10 mM MgCl₂ -10 mM Tris (pH 7.6, or at lower pH whereindicated).

Enzymatic modification of 3 μg RNA by RNase T1 (Pharmacia), RNase T2(BRL), RNase S1 (Pharmacia), or RNase V1 (Pharmacia) was carried out atroom temperature in the presence of 20 μg of carrier tRNA and 10 mMMgCl₂, 10 mM Tris (and 1 mM ZnSO₄ for S1 Nuclease), at pH 7.6 (exceptwhere noted) in a total volume of 40 μL for 10 minutes. Optimal RNaseconcentrations were determined empirically for each batch of RNA.Reactions were stopped by the addition of excess tRNA. The modified RNA(including mock digested RNA not subjected to nucleases) was ethanolprecipitated, and analyzed by primer extension using a ³² P!-labellednegative strand primer and 3 units of avian myeloblastosis virus reversetranscriptase (Life Sciences) at 42° C. (unless otherwise noted) for 30minutes (E. Brown, et al., J. Virol. 65:5828-5838 (1991)). The primersused in these reactions included A-75 (GCCTATAGCCTAGGCAAACG) (SEQ ID NO.5), A-170 (AGAGAAACAGATTTAAGAAC) (SEQ ID NO. 6), A-241(GCCAGAGCCTAGGGCAAGGG) (SEQ ID NO. 7), and A-324 (GTGACGTTCCAAACATCTGT)(SEQ ID NO. 8). Reaction products were separated on a 6% polyacrylamidegel, in parallel with dideoxy sequencing reactions using unmodified RNA.

RNA transcription and transfection. Transcription reactions with SP6 RNApolymerase (Promega) were carried out in 20-μl reaction volumes, with1.25 mM R130 nucleoside triphosphates and 1.5 pg of HaeII-digested DNA,for 90 minutes at 37° C. Immediately upon termination of the reaction,18 μl of the reaction mix was mixed with 30 μl (30 μg) of Lipofectin(BRL), diluted to 100 μl according to the manufacturer's directions, andused to transfect one 60-mm petri dish culture of BS-C-1 or FRhK-4cells. Visual inspection of transcription products in 0.1% sodiumdodecyl sulfate (SDS) agarose gels indicated that the guantity of fulllength 7.5 kb RNA was approximately the same in each transfection.However, in one transfection, RNA products were labelled with traceamounts of ³² p and the resulting 7.5 kb bands excised from the gel andcounted in a scintillation counter. The amount of radioactivity in the7.5 kb bands from different transcription reactions differed by lessthan 20% (results not shown), confirming the validity of the visualquantitation.

For transfections, cells were washed twice with serum-free medium, fedwith 2.5 ml of serum-free medium and the RNA-Lipofectin mixture addeddropwise. Following an overnight incubation, 5 ml of medium containing10% fetal bovine serum were added to each culture. Except where noted,transfections were carried out as direct transfection/radioimmunofocusassays (S. Day, et al., J. Virol. 66:6533-6540 (1992), S. Lemon, L.Binn, and R. Marchwicki, J. Clin. Microbiol. 17:834-839 (1983)). Thus,twenty-four hours after the addition of serum-containing medium, thecells were overlaid with agarose. The cultures were incubated for 7-9days at 31°-32° C., 35.5° C., or 37° C., and processed for detection ofradioimmunofoci as described previously (S. Lemon, L. Binn, and R.Marchwicki, J. Clin. Microbiol. 17:834-839 (1983)).

Where indicated, virus stocks were rescued from transfected FRhK-4 (orBS-C-1) cells maintained without agarose overlays. At harvest, cellswere scraped into 4 ml of medium, and subjected to 3 freeze-thaw cyclesfollowed by brief sonication. Cellular debris was removed by low speedcentrifugation followed by chloroform extraction, and first passagevirus stocks were stored at -70° C. Higher titer (second passage) masterseed stocks were prepared by inoculating 900 cm² roller bottle culturesof confluent FRhK-4 cells with first-passage virus and harvesting asdescribed above after 7 to 9 days of incubation at 35.5° C. (or 31° C.in the case of temperature-sensitive ts! mutants). Working virus (thirdpassage) stocks were recovered by similar passage of the master seedstock in BS-C-1 cells. Virus titers are reported asradioimmunofocus-forming units of virus (rfu) per milliliter (rfu/ml)(S. Lemon, L. Binn, and R. Marchwicki, J. Clin. Microbiol. 17:834-839(1983)).

RNA sequencing. To confirm the presence of mutations in rescued viruses,the genomic RNA of working virus stocks was sequenced in the region ofthe pY1 domain after reverse transcription and amplification of CDNA (nt31 to 317) by an antigen-capture-PCR method (R. Jansen, G. Siegl, and S.Lemon, Proc. Natl. Acad. Sci. USA 87:2867-2871 (1990)). The PCR productwas gel-purified, and used as template in cycle sequencing reactionswith ΔTaq DNA polymerase (U.S. Biochemical Corp.). A negative strandsequencing primer, SLA-229 (GGGGAGAGCCCTGG) (SEQ ID NO. 9), was used inthese reactions. The same strategy was used to sequence hepatitis Avirus strain CP.

One-step growth curve analysis of rescued virus. Approximately 2×10⁵BSC-1 or FRhK-4 cells in individual, replicate wells of a 24-wellculture plate were inoculated at a high multiplicity of infection(range, 2 to 5). At specified time points, supernatant fluids wereremoved from the cultures. The cells were washed twice and lysed by theaddition of 1 ml 0.1% SDS as described previously (S. Day, et al., J.Virol. 66:6533-6540 (1992)). The viral titer of supernatant fluids orcell lysates was subsequently determined by radioimmunofocus assayscarried out in BS-C-1 cells at 35.5° C. (or 31° C. for ts mutants).

Thermostability assay. Normal and ts virus stocks were diluted to 6.8log₁₀ rfu/ml in cell culture medium containing 3% fetal bovine serum,divided into four aliquots, and placed at 0° C. Individual aliquots ofeach virus stock were heated to 50° C., 55° C., or 60° C. for 10 minutesin an automatic thermal cycler. The residual infectious virus titer wasdetermined by radioimmunofocus assay of BS-C-1 cells at 31° C.

II. RESULTS

Secondary structure of the 5' 300 nt of the 5'NTR of HAV. Covariantnucleotide substitutions within the 5'NTRs of different strains of HAVpredict double-stranded helices that are conserved in the secondarystructure of the RNA (E. Brown, et al., J. Virol. 65:5828-5838 (1991)).The presence of numerous covariant substitutions provided a high levelof confidence in predictions of the structure of the 3' half of the5'NTR, but only a single cluster of covariant substitutions (near thetop of stem-loop IIIa) (not shown) has been identified upstream of nt330. Thus previous predictions of the structure in this region of the5'NTR (E. Brown, et al., J. Virol. 65:5828-5838 (1991)) (not shown) werebased almost entirely upon thermodynamic considerations. To test thevalidity of these predictions, we determined the sites at whichsynthetic 5'NTR RNA was susceptible to cleavage by RNases whichpreferentially cleave single-stranded (RNase S1, RNase T1, RNase T2) ordouble-stranded (RNase V1) RNA. The synthetic RNAs utilized in theseexperiments represented the 5' 980 or 1108 nucleotides of the HAV genomeand included 10 additional nucleotides at the 5' terminus which werederived from the vector. These experiments generally confirmed thepredicted secondary structure. Each region within the 5' 303 nts of the5'NTR was examined in at least two separate experiments. The mostprominent single-strand-specific RNAse cleavage sites were locatedprecisely in the predicted loop regions of stem-loops I, IIa, IIb andIIIb, and at the 5' and 3' ends of the extended region flanking the pY1domain (nt 96 to 98 and 135 to 152). The most prominent sites at whichthe double-strand-specific RNase V1 cleaved the RNA were located withinthe stems of stem-loops IIb (nt 81 to 84) and IIId (nt 282 to 285).Other V1 cleavage sites were at nt 74 to 76, in a region betweenstem-loops IIa and IIb which would be base paired in the secondpredicted pseudoknot (not shown).

Surprisingly, RNase V1 cleaved the RNA at multiple sites within the pY1domain, despite previous predictions that this region should be singlestranded. These VI cleavage sites centered on five groups of cytidylicacids that occur as part of the repetitive (U)UUCC(C) motifs, but V1cleavage also occurred at uridylic acids located just downstream of thepY1 domain (nt 141 and 142). Significantly, no single-strand-specificenzymes cleaved the RNA within the region containing the five repetitive(U)UUCC(C) motifs (nt 99 to 130), although relatively strongsingle-strand cleavage sites flanked this domain. These results indicatethat the pY1 domain does not exist as a randomly ordered single-strandedRNA segment, but that it possesses an ordered structure. The V1cleavages in this domain may reflect helical stacking of the RNA, orpossibly noncanonical hemiprotonated C--C base pairing (K. Gehring, J.Leroy, and M. Gueron, Nature 363:561-565 (1993)) (see Discussion). Sincehemiprotonated C--C base pairing is more likely to occur at acidic pH,we carried out V1 digestions over a pH range of between 7.6 and 6.0.There was no enhancement of V1 cleavage at low pH, as might be expectedif C--C base pairing were occurring (data not shown). Parallel analysisof a different region of the 5' NTR confirmed that the enzyme was fullyactive at pH 6.0.

These experiments also provided indirect evidence for the existence ofthe two pseudoknots predicted to involve stem-loops IIa and IIb (notshown). Strong stops for reverse transcriptase were found to occurexactly at the 3' end of these predicted stem-loop structures (notshown). A similar strong stop was not present at the 3' end of the 5'terminal hair-pin (stem-loop I), although a much weaker stop wassometimes observed within a G-C rich region of this stem-loop (nt 26 to28), (not shown). Although it is possible that the helical stems ofstem-loops IIa and IIb are sufficiently stable to inhibit theprogression of reverse transcriptase, the fact that a similar strongstop was not observed at the 3' end of stem loop I, which is longer,more G-C rich, and predicted to have a much lower free energy (notshown) (A. Jacobson, et al., Nucleic Acids Res. 12:45-52 (1984)),suggests that stem loops IIa and IIb are further stabilized by theirinvolvement in pseudoknots.

Deletion mutagenesis of the pyl domain. Although the sequence within thepY1 domain is more variable than that in any other region of the 5'NTR(E. Brown, et al., J. Virol. 65:5828-5838 (1991)), all human hepatovirusstrains studied thus far contain a pyrimidine-rich sequence in thisregion which is 21 to 40 nt in length (see FIG. 4). Each of these virusstrains also preserves the repetitive (U)UUCC(C) motif, although thenumber of these motifs varies from strain to strain. To determinewhether deletion mutations within and flanking the pY1 tract wouldimpair replication, we constructed a full-length cDNA clone with a largedeletion (pΔ99-144) in this domain. This deletion mutant wassubsequently used for construction of additional mutants with smallerdeletions (FIG. 1) (see Materials and Methods). Each of the deletionmutations was confirmed by double-stranded DNA sequencing prior to RNAtranscription and transfection into permissive BS-C-1 or FRhK-4 cells.Results of transfections at 35.5° C. or 31° C. are summarized in FIG. 1.

In direct transfection-radioimmunofocus assays carried out at 35.5° C.,transfection of RNA derived from pP16-pY1, which contains the wild-typeHM175 sequence in the pY1 domain, generated viral replication foci whichwere identical in size to those derived from RNA transcribed from theparental construct, pP35-pY1 (data not shown). However, multipletransfections with pΔ99-144 RNA at standard temperature conditions of35.5° C., in either FRhK-4 or BS-C-1 cells and including two blindpassages of transfected cell harvests, never resulted in recovery ofviable virus (FIG. 1). pΔ99-144 DNA was sequenced completely within themanipulated region (nt 25 to 632). There were no changes from theparental sequence other than the expected 46-nt deletion. To determinewhether a lethal mutation may have occurred elsewhere in the genome, theBspEI/BamHI fragment (nt 25 to 632) from pΔ99-144 was replaced with thecorresponding fragment from the viable mutant pP16-pY1. As expected, RNAfrom the resulting clone generated replication foci that were identicalin size to those of pP16-pY1. Thus, deletion of an extended sequencebetween stem-loops IIb and IIIa (Δ99-144, FIG. 1) resulted in theabsence of successful RNA transfection at physiologic temperature.

RNA derived from cDNA clones with smaller deletions in the pY1 domainproved to be infectious under these conditions (FIG. 3). However, twodifferent replication phenotypes were observed among the rescued viruses(not shown). Viruses rescued from pΔ99-115, pΔ99-130, pΔ99-134,pΔ96-134, pΔ96-137, and pΔ96-139 produced replication foci which weresimilar in size to those of pP16-pY1. Thus, a 44-nt-long deletionmutation which included the entire pY1 domain (Δ96-139) resulted in noapparent impairment of virus replication. In contrast, virus rescuedfrom pΔ131-144 produced very small replication foci in radioimmunofocusassays carried out at 35.5° C. The small replication focus size observedwith this virus prompted an examination of its temperature sensitivity.Parallel titrations of Δ131-144 virus in BS-C-1 cells at 31° C. and 37°C. demonstrated a difference of 1.8 log₁₀ rfu/ml in the titer of theworking virus stock determined in radioimmunofocus assays carried out atthese two temperatures (ts index), confirming that Δ131-144 virus had atemperature-sensitive (ts) replication phenotype (Table 1). In contrast,the ts index of P16-pY1 virus was 0.35±0.08 log₁₀ rfu/ml in multipleassays. Consistent with these results, replication foci of Δ131-144virus were nearly as large as those of P16-pY1 virus at 31° C. (data notshown).

ts Phenotypes of viruses with pY1 deletions extending to nt 140 to 144.Recognition of the ts phenotype of Δ131-144 virus led us to reevaluatethe infectivity of RNA transcribed from pΔ99-144 and pΔ116-144, both ofwhich failed to generate infectious virus in transfections of FRhK-4 orBS-C-1 cells at 35.5° C. (FIG. 1).

                  TABLE 1                                                         ______________________________________                                        TEMPERATURE SENSITIVITY OF 5'NTR DELETION MUTANTS                                    Radioimmunofocus                                                              Size.sup.1                                                             Virus     31° C.                                                                         37° C.                                                                          ts index.sup.2                                                                        Sequencing.sup.3                           ______________________________________                                        P16-pY1   +++     +++      0.35 ± 0.08                                                                        Yes                                        P35-pY1   +++     +++      0.22    Yes                                         99-115   +++     +++      0.11    Yes                                         99-130   +++     +++      0.54    Yes                                         99-134   +++     +++      0.21    Yes                                         96-134   +++     +++      n.d..sup.4                                                                            Yes                                         96-137   +++     +++      0.29 ± 0.04                                                                        Yes                                         96-139   +++     +++      0.40    n.d.                                        96-140   ++(+)   +        0.73 ± 0.17                                                                        n.d.                                        96-141   ++(+)   +        >1.40   Yes                                         99-144   ++      (+)      3.60    Yes                                        116-144   ++(+)   (+)      1.90    Yes                                        131-144   ++(+)   +        1.80    Yes                                        ______________________________________                                         .sup.1 The relative sizes of replication foci were scored subjectively:       +++, equivalent to parental P16pY1 virus; ++(+), occasionally equivalent      to P16pY1 but tended to be smaller; ++, almost always smaller than P16pY1     +, small foci but always apparent; (+), tiny foci not always apparent in      radioimmunofocus assays.                                                      .sup.2 ts index = log.sub.10  titer 31° C.! - log.sub.10  titer        37° C.! in radioimmunofocus assays carried out in BSC-1 cells, S.E     where 3 or more assays were carried out. The 96139 result is a mean of tw     assays, and the 96141 result a mean of 3 assays (see Results).                .sup.3 Mutation confirmed by RNA sequencing of rescued virus.                 .sup.4 n.d. = not done.                                                  

Repeat RNA transfections of FRhK-4 cells at 31° C. resulted in therescue of viruses with marked ts phenotypes (not shown). The ts index ofΔ99-144 virus was 3.6 log₁₀ rfu/ml, while that of Δ116-144 virus was 1.9log₁₀ rfu/ml (Table 1). Because the ts indices of the Δ96-137 andΔ96-139 viruses were 0.29±0.04 and 0.40 log₁₀ rfu/ml respectively,similar to that of the parent P16-pY1 virus (Table 1), these resultssuggested that the 3' extension of the deletion to include nt 140 to 144was responsible for the ts replication phenotype. Interestingly,although deletion of the region spanning nt 99 to 130 (Δ99-130 andΔ99-134 viruses, Table 1) had no significant impact on virus replicationat 37° C., the deletion of this region in association with the deletionof nt 131 to 144 resulted in a significant enhancement of the tsphenotype (compare the ts indices of the Δ99-144 and Δ131-144 viruses,3.6 versus 1.8 log₁₀ rfu/ml, respectively, Table 1). Because the tsindex of the Δ116-144 virus was only 1.9, this enhancement of the tsphenotype was due primarily to deletion of the highly conserved first2.5 (U)UUCC(C) motifs located between nt 99 and 115.

In order to define more precisely the nucleotide deletions responsiblefor the ts phenotype, two additional mutant cDNA clones wereconstructed, pΔ96-140 and pΔ96-141. RNA transfections at 35.5° C.produced viruses with moderate ts phenotypes. The ts index of Δ96-140virus was 0.73±0.17 log₁₀ rfu/ml, greater than that of the parent virusP16-pY1 (0.35±0.08 log₁₀ rfu/ml) (Table 1). The ts index of Δ96-141virus was >1.4 log₁₀ rfu/ml (1.5, >1.12, and >1.5 log₁₀ rfu/ml in threeseparate experiments). Thus, progressively greater ts indices wereobserved with viruses in which the pY1 deletion mutations extended in a3' direction into the sequence spaning nt 140 to 144 (GUUGU). However,we do not yet know whether deletion of this sequence alone confers thets phenotype. Although these ts viruses replicated much more efficientlyat the permissive temperature, the replication foci of viruses with verylarge deletions (Δ96-141 and Δ99-144) were smaller than those of non-tsviruses (e.g. Δ96-137) at 31° C.

Double-stranded DNA sequencing of the cDNA region (nt 25 to 632)manipulated during mutagenesis of two of the ts cDNA clones (pΔ131-144and pΔ116-144) documented only the expected deletion mutations.Replacement of this segment in the non-ts pP16-pY1 clone with thecorresponding segment from pΔ131-144 conferred the ts phenotype on theproduct virus, confirming that the reduced replication capacity at 37°C. was due to the engineered deletion and not to an adventitiousmutation elsewhere in the genome. Equally important, the expecteddeletions were confirmed in the RNA sequence of each of the rescuedviruses (except Δ96-139 and Δ96-140, which were not sequenced) byantigen-capture-PCR of virus, followed by double-stranded DNA sequencingof the amplified product (Table 1). In no case was there reason tosuspect that any of the rescued virus stocks had developed revertant orpseudorevertant mutations to compensate for the engineered deletions,since replication foci were numerous and similar in size on primarypassage in direct transfection/radioimmunofocus assays.

The phenotype of individual mutants was the same following successfultransfection of either BS-C-1 or FRhK-4 cells. BS-C-1 cells wereconsistently more difficult to transfect, but the replication foci ofeach of the rescued viruses was larger in BS-C-1 cells than in paralleltransfections carried out in FRhK-4 cells (data not shown). Thisobservation is consistent with the fact that each of these virusescontains cell-culture adaptation mutations at nt 152 and 203 to 204which have been shown to promote replication of the virus in BS-C-1 butnot FRhK-4 cells (S. Day, et al., J. Virol. 66:6533-6540 (1992)).

The phenotypes of the rescued viruses remained stable for up to fourpassages as judged by the size of replication foci in radioimmunofocusassays. Further evidence for the stability of the ts phenotype wasprovided by experiments in which BS-C-1 cells infected with ts variants(Δ99-144 and Δ96-141 virus) were maintained for up to 3 weeks at thenonpermissive temperature (37° C.), after an initial 24-hour incubationat the permissive temperature (31° C). Virus harvests prepared fromthese cells were subsequently tested in radioimmunofocus assays at thenonpermissive temperature in order to detect large focus revertants. Nosuch revertants were isolated (data not shown).

Analysis of ts virus replication under one-step growth conditions.Although radioimmunofocus size is an accurate measure of the replicationefficiency of HAV in cultured cells (S. Day, et al., J. Virol.66:6533-6540 (1992)), BS-C-1 and FRhK-4 cells were infected underone-step growth conditions in order to quantitate better differences inthe kinetics of replication of different deletion mutants. At thepermissive temperature (31° C.) in BS-C-1 cells, the replication ofΔ131-144 virus (ts index, 1.8) was somewhat delayed compared withreplication of the parental P16-pY1 virus or the large deletion mutantΔ96-137 (FIG. 2A-C). The latter two viruses demonstrated similarreplication kinetics, with virus yields approaching maximum by 72 hourspostinoculation. In contrast, maximum yields of Δ131-144 were notreached until 144 hours postinoculation. This difference in replicationkinetics was reflected also in the somewhat smaller size of Δ131-144replication foci at 31° C. The higher intracellular virus titerimmediately after adsorption of Δ131-144 (time 0, FIG. 2A-C) likelyreflects a higher multiplicity of infection in cells inoculated with theΔ131-144 virus.

At the nonpermissive temperature (37° C.), replication of Δ131-144 viruswas further delayed, with no increase over input virus noted until after72 hours postinoculation. Between 72 and 216 hours, the increase in thetiter of Δ131-144 virus paralleled that observed between 18 and 72 hoursat the permissive temperature (FIG. 2A-C). In contrast, there was nodifference in the growth kinetics of P16-pY1 and Δ96-137 viruses at 31°C. and 37° C., consistent with the low ts indices of these viruses(Table 1). The fact that the rate of intracellular accumulation ofΔ131-144 virus between 72 and 216 hours at the nonpermissive temperatureparalleled the rate of accumulation between 12 and 150 hours at thepermissive temperature suggests that the ts phenotype of Δ131-144 mightbe due to a temperature-sensitive step occuring relatively early in thevirus replication cycle. Additional one-step growth experimentsconfirmed that the replication of Δ131-144 virus was significantlydelayed in comparison with P16-pY1 and Δ100-131 viruses at 35.5° C. inboth FRhK-4 and BS-C-1 cells (data not shown). In general, in theseone-step growth experiments, the final virus yield obtained with the tsΔ131-144 virus was similar to that obtained with the non-ts viruses.

Contribution of P2 region mutations to the ts phenotype. All of thedeletion mutants described above were constructed in a background whichincluded the P2 genomic region of the rapidly replicating, cytopathicstrain, HM175/18f (see Methods). Thus, it was possible that the tsphenotype of the mutants described above might be derived in part fromone or more of the numerous mutations present in the P2 region (S.Lemon, et al., J. Virol. 65:2056-2065 (1991)). To address thispossibility, the P2 region from the cell culture-adapted HM175/P35variant (pHAV/7) (J. Cohen, et al., J. Virol. 61:3035-3039 (1987)) wasreintroduced into the ts CDNA clone pΔ131-144 to producepΔ131-144/P2P35. Virus rescued from pΔ131-144/P2P35 RNA demonstrated ats phenotype similar to Δ131-144 virus (data not shown), indicating thatthe ts phenotype was not codependent upon the presence of HM175/18f P2region mutations. However, as expected, this virus replicated much moreslowly than Δ131-144, requiring 2-3 weeks for demonstration ofreplication foci following RNA transfection, even at the permissivetemperature.

Thermostability of ts virus particles. We compared the thermostabilityof the Δ131-144 virus with that of the P16-pY1 parent in order todetermine whether the reduction in titer of this ts strain at thenonpermissive temperature might reflect increased thermolability ofvirions due to altered interactions between capsid proteins and genomicRNA. The infectious titers of the P16-pY1 and Δ131-144 viruses werereduced to a similar extent following brief incubation at temperaturesranging from 50° to 60° C. (FIG. 2A-C). Thus the ts phenotype ofΔ131-144 virus is not related to reduced thermostability of the virus.

Deletion mutation involving stem-loop IIb. All of the deletion mutationsdescribed above were located between stem-loop structures predicted toflank the pY1 region. To determine the impact of extension of thesedeletions in a 5' fashion into stem-loop IIb, an additional cDNA mutant(pΔ93-134) was constructed. Compared with the viable pΔ96-134 mutant,the deletion mutation in pΔ93-134 extends in a 5' direction by anadditional 3 nt and includes the 3' terminal 2 nt of stem-loop(pseudoknot) IIb (FIG. 1). Multiple transfections of FRhK-4 or BS-C-1cells with RNA derived from pΔ93-134, at either 31° C. or 35.5° C.failed to yield infectious virus (FIG. 1). In addition, aserendipitously discovered second-site cDNA mutant derived from theviable pΔ99-134 mutant which had an additional, random mutationinvolving a G-to-U substitution at nt 85, also failed to produceinfectious virus after RNA transfection (data not shown). The G-to-Usubstitution at nt 85 would be predicted to destabilize the putativepseudoknot involving stem-loop IIb. These data suggest that retention ofthe secondary and possibly tertiary RNA structure in this region of the5'INTR is essential for infectivity of the virus and provide furtherindirect support for the proposed structural model.

III. DISCUSSION

Virus mutants with deletions in the pY1 region which were rescued fromtransfected cells demonstrated two distinctly different replicationphenotypes. Five mutant viruses with deletions ranging from 14 to 46nucleotides in length and extending into the critical domain of nt 140to 144 (Δ99 -144, Δ116-144, Δ131-144, Δ96-140, and Δ96-141) were foundto have a ts replication phenotype. If the entire sequence between nt140 and nt 144 was removed, the resulting viruses were strongly ts(Δ99-144, Δ116-144, and Δ131-144). These viruses demonstrated areduction in viral titer at the nonpermissive temperature (ts index)ranging from 1.8 to 3.6 log₁₀ rfu/ml (Table 1). In contrast, a secondgroup of mutant viruses with equally large deletions, up to 44nucleotides in length, but not involving nt 140 to 144, replicated asefficiently as the parental virus at 37° C. and 31° C. These dataindicate that the pY1 domain (nt 99 to 138) of HM175 virus is notrequired for replication in cultured cells, while the flankingsingle-stranded domain (nt 140 to 144) is essential for efficientreplication at physiological temperatures. The marked difference betweenthe ts index of the Δ99-144 mutant and those of the Δ116-144 andΔ131-144 mutants (3.6 versus 1.9 and 1.8 log₀ rfu/ml, respectively,Table 1) demonstrates that the additional deletion of sequence elementswithin the pY1 domain (particularly nt 99 to 115) substantially enhancesthe ts phenotype of virus lacking nt 131 to 144. The critical ts domain(CUUGU, nt 140 to 144) is located at the 3' end of the pY1 tract. Thesenucleotides are part of a larger single-stranded segment which isaccessible on the surface of the tertiary structure of the folded RNA,as evidenced by the ability of single-strand-specific RNases to cleavewithin the sequence spanning nt 135 to 152 (not shown). Although it isapparently not involved in base-pairing interactions with other regionsof the 5'NTR, this short segment, especially the trinucleotide sequenceUGU (nt 142 to 144), is very well conserved among different hepatovirusstrains.

Large pY1 tract deletions which did not involve the critical nt 140 to144 domain had no apparent effect on the replication of virus in FRhK-4or BS-C-1 cell cultures maintained at physiologic temperatures (Table1). Note the presence of smaller pY1 deletions in the sequences of otherhepatovirus strains (FIG. 4). The sequence of several of these strains,MBB (A. Paul, et al., Virus Res. 8:153-171 (1987)), CF53 and PA21 (E.Brown, et al., J. Virol. 65:5828-5838 (1991)) was obtained from RNAisolated from cell culture-adapted variants, and thus these deletionsmay have occurred during adaption and passage in cultured cells. Asshown in FIG. 1, a 4-nt deletion (nt 131 to 134) is known to haveoccurred in this domain during adaptation and passage of the HM175strain in cell culture (J. Cohen, et al., Proc. Natl. Acad. Sci. USA84:2497-2501 (1987)).

The foregoing is illustrative of the present invention, and not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

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NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCCTATAGCCTAGGCAAACG20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGAGAAACAGATTTAAGAAC20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCCAGAGCCTAGGGCAAGGG20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTGACGTTCCAAACATCTGT20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGGGAGAGCCCTGG14                                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      UAGGCUAAAUUUUCCCUUUCCCUUUUCCCUUUCCUAUUCCCUUUGUUUUGCUUGUAAAUA60                UUAAUUCCUGC71                                                                 (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      UAGGCUAAAUUUUCCCUUUCCCUUUUCCCUUUCCUAUUCCCUUUGUUUUGCUUGUAAAUA60                UUGAUUCCUGC71                                                                 (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      UAGGCUAAAUUUUCCCUUUCCCUUUUCCCUUUCCAAUUCCCUUUUGCUUGUAAAUAUUGA60                UUCCUGC67                                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      UAGGCUAAACCCUUUCCUAUUCCCUUUGUUUUGCUUGUAAAUAUUGAUUCCUGC54                      (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      UAGGCUAAAUUUGUUUUGCUUGUAAAUAUUGAUUCCUGC39                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      UAGGCUAAAUUUUGCUUGUAAAUAUUGAUUCCUGC35                                         (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      UAGGCUUUUUGCUUGUAAAUAUUGAUUCCUGC32                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      UAGUUUUGCUUGUAAAUAUUGAUUCCUGC29                                               (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      UAGGCUUGCUUGUAAAUAUUGAUUCCUGC29                                               (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      UAGGCUCUUGUAAAUAUUGAUUCCUGC27                                                 (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      UAGGCUUUGUAAAUAUUGAUUCCUGC26                                                  (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      UAGGCUUGUAAAUAUUGAUUCCUGC25                                                   (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      UAGGCUAAAAAAUAUUGAUUCCUGC25                                                   (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      UAGGCUAAAUUUUCCCUUUCCCUUUAAAUAUUGAUUCCUGC41                                   (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      UAGGCUAAAUUUUCCCUUUCCCUUUUCCCUUUCCUAUUCCCAAAUAUUGAUUCCUGC57                   (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      UAGGCUAAAUUUUCCCUUUCCCUUUUCCCUUUCCUAUUCCCUUUGUUUUGCUUGUAAAUA60                UUAAUU66                                                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      UAGGCUAAAUUUCCCUUUCCCUGUCCUUCCCCUAUUUCCCUUUGUUUUGCUUGUAUAUAU60                UAAUU65                                                                       (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      UAGGCUAAAUUUCCCUUUCCCUGUCCUUCCCCUAUUUCCCUUUGUUUUGUUUGUAAAUAU60                UAAUU65                                                                       (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      UAGGCUAAAUUUCCCUUUCCCUGUCCCUUCCCUAUUUCCCUUGUUUUAUUUGUAAAUAUU60                AAUU64                                                                        (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      UAGGCUAAAUUUCCCUUUCCCUGUCCUUCCCCUAUUUACCUUUGUUUUGCUUGUAUAUAU60                UAAUU65                                                                       (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      UAGGCUAAAUUUCCCUUUCCCUGUCCCUUCCCUAUUUCCCUUUAUUUGCUUGUAAAUAUU60                AAUU64                                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      UAGGCUAAAUUUCCCUUUCCCUGUCCUUCCCUUAUUUCCCUUUGUUUUGCUUGUAAAUAU60                UGAUU65                                                                       (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      UAGGCUAAAUUUCCCUUUCCCUGUCCCUCCCUUAUUUCCCUUUGUUUUGCUUGUAAAUAU60                UAAUU65                                                                       (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      UAGGCUAAAUUUUCCCUUUCCCCUUCCCCUUCCUUGUUUUGAUUGUAAAUAUUAAUU57                   (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      UAGGCUAAAUUUCCCUUUUUCCCUUUCCCUUUAUUGUUGUAAAUAUUAAUU51                         (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      UAGGCUAUUUCUCCCCUUCCCUUUUCCCUGUUUUGUGUAAAUAUUAAUU49                           (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      UAGGCUAAUUUUCCCUUUUCCUUUUCCCUGUGUUAUUGUAAAUAUUAAUU50                          (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      UAGGCUAAAUUUCCCUUUUCCCUUUCCCUUUAAUGUUGUAAAUAUUGAUU50                          __________________________________________________________________________

That which is claimed is:
 1. A live hepatitis A virus deletion mutanthaving a deletion mutation in the 5' nontranslated region of the viralgenome, wherein said deletion mutation is a pY1 deletion mutation thatcauses said virus to retain the ability to replicate in monkey kidneycells, and wherein said pY1 deletion mutation is selected from the goupconsisting of:(a) Δ99-134 deletion mutants; (b) Δ96-134 deletionmutants; (c) Δ96-137 deletion mutants: (d) Δ96-139 deletion mutants, and(e) Δ96-140 deletion mutants.
 2. A virus according to claim 1, whereinsaid deletion mutation is an attenuating mutation.
 3. A cDNA encoding avirus according to claim
 1. 4. A method for inducing protective immunityagainst hepatitis A virus in a subject, comprising:administering to saidsubject an infectious, immunogenic, hepatitis A virus carrying anattenuating mutation, said virus being administered in an amounteffective to induce protective immunity against hepatitis A virus, andsaid attenuating mutation being a deletion mutation in the 5'nontranslated region of the viral genome, wherein said deletion mutationis a pY1 deletion mutation that attenuates said virus, and wherein saidpY1 deletion mutation is selected from the group consisting of: (a)Δ99-134 deletion mutants; (b) Δ96-134 deletion mutants; (c) Δ96-137deletion mutants; (d) Δ96-139 deletion mutants, and (e) Δ96-140 deletionmutants.
 5. A method according to claim 4, wherein said administeringstep is carried out by orally administering said virus to said subject.6. A method according to claim 4, wherein said administering step iscarried out by parenterally injecting said virus into said subject.
 7. Avaccine formulation useful for inducing protective immunity againsthepatitis A virus, comprising, in a pharmaceutically acceptable carrier,an infectious, immunogenic, hepatitis A virus carrying an attenuatingmutation in an amount effective to induce protective immunity againsthepatitis A virus, said attenuating mutation being a deletion mutationin the 5' nontranslated region of the viral genome that is a pY1deletion mutation that attenuates said virus, wherein said pY1 deletionmutation is selected from the group consisting of:(a) Δ99-134 deletionmutants; (b) Δ96-134 deletion mutants; (c) Δ96-137 deletion mutants; (d)Δ96-139 deletion mutants; and (e) Δ96-140 deletion mutants.
 8. A vaccineformulation according to claim 7, wherein said vaccine formulation is anoral vaccine formulation.
 9. A vaccine formulation according to claim 7,wherein said vaccine formulation is a parenterally injectable vaccineformulation.
 10. A vaccine formulation according to claim 7, whereinsaid vaccine formulation is an inhalation vaccine formulation.